Central neuropathic pain (CNP) is a debilitating condition that affects about 70% of patients after spinal cord injury (SCI). We have shown that chronic astrocytic activation contributes to the development and maintenance of CNP after SCI in rats (CNP rats; Nesic et al., 2005). However, the mechanisms underlying astrocytes' roles in CNP remain unknown. Reactive astrocytes in chronically injured spinal cords of CNP rats express significantly higher levels of the water channel protein Aquaporin 4 (AQP4; Nesic et al., 2005, 2006). Altered AQP4 levels are always associated with pathological changes, and nearly always contribute to edema in different CNS pathologies. Our central hypothesis is that excessive upregulation of AQP4 in chronically injured spinal cords induces water accumulation within astrocytes, and astrocytic swelling. Numerous processes are triggered by this swelling that can affect neuronal excitability and thus pain development: ion disturbance (e.g. K+ buffering), amino acid release, Ca2+ signaling and neurotransmission changes. Additionally, we hypothesize that glial swelling counterbalanced by extracellular space (ECS) volume shrinkage, causes ECS hypertonicity. We hypothesize that ECS hypertonicity will trigger upregulation of Aquaporin 1 (AQP1) in the neurons and pain fibers of Laminae I and II. It has been shown that AQP1 is important for normal pain processing in mouse dorsal horns. We hypothesize that small interfering RNA specific for rat AQP4 (AQP4 siRNA) will block persistent up-regulation of AQP4 in chronically injured spinal cords. Our goal is to test the ability of AQP4 siRNA to alleviate CNP after SCI: (a) by reducing astrocytic swelling, and (b) by decreasing AQP1 expression in pain fibers. We will first establish optimal AQP4 siRNA administered intrathecally for 2 weeks (day 21-35 after SCI) using AQP4 Western blot analyses; and than test the effect of AQP4 siRNA on: (1) CNP (forelimb, trunk and hindlimb mechanical allodynia); (2) astrocytic swelling in chronically injured spinal cords using electron microscopy and by measuring water content; (3) AQP1 expression levels in Laminae I and II, using immunohistochemistry, and (4) test the effect of AQP4 siRNA on astrocytic activation by measuring GFAP levels using Western blots and immunohistochemistry. Our hypothesis on the involvement of AQP4, AQP1 and impaired osmoregulation in the pathogenesis of neuropathic pain conditions after SCI is novel and has not been previously studied.Health relevance: Central neuropathic pain (CNP) is a debilitating condition that affects about 70% of patients after spinal cord injury (SCI). Only a small percentage of patients with CNP get even partial relief from current drugs that target neurons. We expect that our results should lay the foundation for an important new therapeutic approach targeting osmoregulation of astrocytes to attenuate CNP after SCI. [unreadable] [unreadable] [unreadable]